Paracrine crosstalk between endothelial cells and cardiomyocytes during inflammation
File(s)
Author(s)
Fourre, Jerome
Type
Thesis
Abstract
Endothelial cells (EC) release paracrine factors which can modulate the survival, morphology, and function of neighbouring cardiomyocytes (CM), e.g. contraction and relaxation. In multiple cardiomyopathies, as well as in heart failure, EC dysfunction correlates strongly with severity and prognosis, and moreover, endothelial inflammation is thought to precede this dysfunction. While it is known that EC functions are modified by inflammation, the paracrine effects of this response on neighbouring CM are poorly understood and too often confused with those of endothelial dysfunction. I hypothesised that calcium handling and contractile properties of CM were differently regulated by inflamed EC.
To investigate this, a co-culture system was first validated as a model of the paracrine EC-CM crosstalk. A pro-inflammatory cocktail of TNFα, IL-1β and hIL-6 (“Cytomix”) was then validated as a pre-conditioning treatment for cardiac microvascular EC. Adult ventricular CM were co-cultured with untreated or Cytomix-treated EC. Calcium transients were then analysed in CM using Fluo-4 and Fura-2. Cell contractility and relaxation were also studied using an automated CytoCypher™ system. Finally, living rat myocardial slices were used to investigate the effects of Cytomix in a more complex heterocellular model.
Co-culture with Cytomix-treated EC induced a significant shortening of calcium transients in CM compared to untreated EC (thus validating the hypothesis). This was due to an increase in SERCA activity. However, the cell shortening amplitude and rate of relaxation were unaffected by co-culture or Cytomix treatment, while data also suggests that the myofilament sensitivity to calcium was unchanged. In cultured myocardial slices, Cytomix treatment increased force but not the passive tension, although this was limited to high levels of stretch and kinetics were unchanged compared to untreated slices.
Further work is required to better define and understand these mechanisms. This could help determine the therapeutic potential of controlling EC function in inflammatory heart diseases.
To investigate this, a co-culture system was first validated as a model of the paracrine EC-CM crosstalk. A pro-inflammatory cocktail of TNFα, IL-1β and hIL-6 (“Cytomix”) was then validated as a pre-conditioning treatment for cardiac microvascular EC. Adult ventricular CM were co-cultured with untreated or Cytomix-treated EC. Calcium transients were then analysed in CM using Fluo-4 and Fura-2. Cell contractility and relaxation were also studied using an automated CytoCypher™ system. Finally, living rat myocardial slices were used to investigate the effects of Cytomix in a more complex heterocellular model.
Co-culture with Cytomix-treated EC induced a significant shortening of calcium transients in CM compared to untreated EC (thus validating the hypothesis). This was due to an increase in SERCA activity. However, the cell shortening amplitude and rate of relaxation were unaffected by co-culture or Cytomix treatment, while data also suggests that the myofilament sensitivity to calcium was unchanged. In cultured myocardial slices, Cytomix treatment increased force but not the passive tension, although this was limited to high levels of stretch and kinetics were unchanged compared to untreated slices.
Further work is required to better define and understand these mechanisms. This could help determine the therapeutic potential of controlling EC function in inflammatory heart diseases.
Version
Open Access
Date Issued
2020-11
Date Awarded
2021-06
Copyright Statement
Creative Commons Attribution NonCommercial NoDerivatives Licence
Advisor
Mason, Justin
Terracciano, Cesare
Sponsor
British Heart Foundation
Grant Number
FS/15/65/32036
Publisher Department
National Heart and Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)